屡配不孕母牛FOXP3、FSHR、FMR1基因多态性与生殖激素相关性分析

doi:10.11843/j.issn.0366-6964.2024.06.043

摘要/Abstract

摘要：

旨在研究叉头框蛋白P3基因(forkhead box protein P3，FOXP3)、促卵泡激素受体基因(follicle stimulating hormone receptor，FSHR)、智力低下1基因(fragile X mental retardation 1，FMR1)的多态性，并探讨了基因多态性对母牛屡配不孕以及母牛激素水平的影响。通过PCR方法扩增基因突变位点的序列，对PCR结果进行酶切鉴定和测序峰图分析，统计三种基因的基因型。分析三种基因突变的组间差异。使用ELISA法检测两组雌激素(estrogen，E)、促卵泡激素(follicle-stimulating hormone，FSH)、催乳素(prolactin，PRL)、睾酮素(testosterone，T)、促黄体生成素(luteinizing hormone，LH)激素浓度。并将基因多态性与激素水平进行关联分析。FOXP3基因检测出3种基因型：AA、AG和GG型；FSHR基因检测出3种基因型：AA、AC和CC型；FMR1基因5'UTR端302 bp处检测到T碱基插入。卡方适应性检验结果表明，FOXP3基因g.92, 377, 635 A>G位点、FSHR基因g.31, 450, 279 A>C位点均低于哈代-温伯格平衡状态(P＜0.05)。关联性分析表明，FOXP3基因G等位基因、FSHR基因C等位基因与母牛屡配不孕显著正相关性。屡配不孕组E、FSH、PRL、T激素与健康母牛组差异显著(P < 0.05)。FOXP3基因AA、GG基因型E激素浓度与AG基因型差异显著(P < 0.05)；FSHR基因AA、AC基因型E激素浓度与CC基因型差异显著(P < 0.05)。FOXP3、FSHR基因在西门塔尔牛、褐牛及安格斯牛群体中具有多态性，这说明基因可能是影响母牛屡配不孕的潜在遗传因素。其中，FOXP3基因G等位基因在健康组与屡配不孕组中差异显著(P < 0.05)，FSHR基因C等位基因在健康组与屡配不孕组中差异显著(P < 0.05)。FOXP3基因的g.92, 377, 635 A>G位点G等位基因与母牛患屡配不孕疾病呈显著正相关(P < 0.05，r=0.16)，FSHR基因的g.31, 450, 279 A>C位点C等位基因与母牛屡配不孕具有显著正相关性(P < 0.05，r=0.14)。FOXP3以及FSHR基因的多态性位点可能作为母牛屡配不孕的标志性识别位点，可用于筛选屡配不孕母牛的潜在候选遗传标记，这需要进一步的证明。FOXP3基因G等位基因与E浓度呈弱正相关(P < 0.05，r=0.16)，FMR1基因T碱基的插入与E浓度呈弱正相关(P < 0.05，r=0.187)。然而FOXP3、FSHR基因的变异如何调控母牛繁殖能力还需进一步研究证实。

关键词: 牛, 屡配不孕, 基因多态性, 激素, 关联分析

Abstract:

The polymorphisms of forkhead box protein P3 (FOXP3), follicle stimulating hormone receptor (FSHR) and fragile X mental retardation 1 (FMR1) were studied, and the effects of gene polymorphisms on infertility and hormone levels in cows were discussed. The sequence of the gene mutation site was amplified by PCR, and the PCR results were identified by enzyme digestion and sequencing peak analysis, and the genotypes of the three genes were counted. The intergroup differences in the mutations of the three genes were analyzed. The concentrations of estrogen (E), follicle-stimulating hormone (FSH), prolactin (PRL), testosterone (T) and luteinizing hormone (LH) in the two groups were detected by ELISA. The correlation between gene polymorphism and hormone level was analyzed. Three genotypes of FOXP3 gene were detected: AA, AG and GG; three genotypes of FSHR gene were detected: AA, AC and CC; T base insertion was detected at 302 bp of 5'UTR of FMR1 gene. The results of chi-square adaptability test showed that the 92 377 635 A>G loci of FOXP3 gene and the 31 450 279 A>C loci of FSHR gene were lower than Hardy-Weinberg equilibrium (P < 0.05). Correlation analysis showed that FOXP3 gene G allele and FSHR gene C allele were significantly positively correlated with repeated infertility in cows. There were significant differences in E, FSH, PRL and T hormones between the infertile group and the healthy mother group (P < 0.05). The E hormone concentration of FOXP3 AA and GG genotypes was significantly different from that of AG genotype (P < 0.05). FSHR gene AA, AC genotype E hormone concentration was significantly different from CC genotype (P < 0.05). (concentration) was significantly different from AG genotype (P < 0.05); FSHR gene 31 450 279 A>C locus AA, AC genotype E hormone content (concentration) FOXP3 gene and FSHR gene are polymorphic in Simmental, Brown and Angus populations, which indicates that the gene may be a potential genetic factor affecting the infertility of cows. The G allele of FOXP3 gene was significantly different between the healthy group and the multiple infertility group (P < 0.05), and the C allele of FSHR gene was significantly different between the healthy group and the multiple infertility group (P < 0.05). The G allele of g.92 377 635 A>G locus of FOXP3 gene was significantly positively correlated with multiple infertility in cows (the C allele of PC locus was significantly positively correlated with multiple infertility in cows (P < 0.05, r=0.14).The polymorphism sites of FOXP3 gene and FSHR gene may be used as the marker identification sites of repeated mating infertility in cows, which can be used to screen the potential candidate genetic markers of repeated mating infertility cows, which needs further proof. The G allele of FOXP3 gene was weakly positively correlated with E concentration (P < 0.05, r=0.16), and the T base insertion of FMR1 gene was weakly positively correlated with E concentration (P < 0.05, r=0.187). However, how the variation of FOXP3 gene and FSHR gene regulates the reproductive ability of cows needs further study.

Key words: cattle, infertility cows, gene polymorphism, hormone, relevance analysis

中图分类号:

S857.22

王选艺, 孙亚伟, 龙雨薇, 王俪颖, 周渝新, 李娜, 马雪连, 赵红琼, 姚刚. 屡配不孕母牛FOXP3、FSHR、FMR1基因多态性与生殖激素相关性分析[J]. 畜牧兽医学报, 2024, 55(6): 2727-2740.

Xuanyi WANG, Yawei SUN, Yuwei LONG, Liying WANG, Yuxin ZHOU, Na LI, Xuelian MA, Hongqiong ZHAO, Gang YAO. Correlation Analysis of FOXP3, FSHR, FMR1 Gene Polymorphisms and Reproductive Hormones in Infertile Cows[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2727-2740.

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基因 Gene	登录号 NCBI ID	引物序列(5′→3′) Primers sequences	退火温度/℃ Annealing temperature	产物长度/bp Product length
FMR1	507482	F: 5′-ACGCGGTTTCACTGTTTACAC-3′	49.8	310
		R: 5′-ACCTTGTAGAAAGCGCCATTG-3′
FOXP3	506053	F: 5′-ATTCTAAGGTTCTGGCATCGT-3′	60.3	618
		R: 5′-GCCAGAGCATATAAGAAACCACT-3′
FSHR	281172	F: 5′-CTGCCTCCCTCAAGGTGCCCCTC-3′	58.0	306
		R: 5′-AGTTCTTGGCTAAATGTCTTAGGGGG

品种 Variety	基因型频率 Gene frequency			基因频率 Genotype frequency		χ²	P值 P value
品种 Variety	AA	AG	GG	A	G	χ²	P值 P value
西门塔尔牛Simmental heifer	0.70	0.27	0.03	0.84	0.16	0.826	0.363
褐牛Brown cattle	0.62	0.30	0.08	0.77	0.23	0.241	0.623
安格斯牛Aberdeen angus	0.55	0.36	0.09	0.73	0.27	5.046	0.025^*

基因Gene	遗传纯和度He	杂合度Ho	有效等位基因数Ne	多态信息含量PIC
FOXP3	0.624	0.376	1.60	0.33

品种 Variety	基因型频率 Gene frequency			基因频率 Genotype frequency		χ²	P值 P value
品种 Variety	AA	AC	CC	A	C	χ²	P值 P value
西门塔尔牛Simmental heifer	0.52	0.35	0.13	0.70	0.30	2.267	0.132
褐牛Brown cattle	0.66	0.30	0.04	0.81	0.19	0.584	0.445
安格斯牛Aberdeen angus	0.75	0.21	0.04	0.86	0.14	7.198	0.007^**

基因Gene	遗传纯和度He	杂合度Ho	有效等位基因数Ne	多态信息含量PIC
FSHR	0.621	0.379	1.61	0.35